Apparatus and method to measure platelet contractility

ABSTRACT

An apparatus and method for measuring blood platelet contractility, hereinafter called a “retractometer” is disclosed. Also disclosed is a system apparatus and method for automatically measuring platelet contractility in a plurality of samples, having an array of retractometer units and an electronic solenoid valve controller to fully automate screening in clinical and research situations and save costs in labor.

This work was supported by National Institute of Health Grant No.R29-HL57430-01.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally concerns blood clotting components andmechanisms.

The present invention particularly concerns new devices and methods forstudying clotting mechanisms and factors. More specifically, anapparatus and method for measuring and monitoring health and activity ofplatelets and other clotting factors are described. Most specifically, aclot retractometer and its method of use are provided to measure clotcontractility forces as a means to provide a single point “funneldetection” procedure useful in aiding physiological and clinicalresearch and patient diagnosis and monitoring of many diseases, as wellas screening populations.

2. Description of Related Art

Introduction

Everyone has seen a clot form as a result of injury to tissue, such as,for example, a scrape, puncture or a bleeding nose. However, theformation of a clot is a complex, cascading process that is still notcompletely elucidated, either physiologically or clinically. Theclotting phenomenon, or lack thereof, is manifest in numerous clinicalconditions, and is relevant to their prognosis.

In response to soft tissue injury, the haemostatic mechanism isactivated to stop bleeding and restore vascular integrity. Blood proteinand cellular interactions lead to the formation of a platelet plug andultimately generation of clot comprising platelets and protein fibers.These reactions have to occur rapidly because the amount of blood lostis dependent on the time required to arrest the bleeding. Although rapidstoppage of blood loss is critical in some cases, inappropriateinduction of clotting can have devastating effects such as decreasedblood flow to the organs and resultant ischemic damage, such as heartattacks and stroke if the clot is not solubilised. To counterbalancethese potentially severe consequences the haemostatic system is usescertain clotting inhibitors, and clot-dissolving enzymes.

Following vascular damage, the exposure of flowing platelets to thesubendothelial lining allows the establishment of adhesive interactionswith the immobilized surfaces. Platelets then become activated due tocontact with thrombogenic substrates and stimulation by locally releasedor generated agonists. Subsequent platelet deposition relies on thebinding of plasma-soluble adhesive molecules, and on the externalizationof adhesive molecules from the platelets' granular reservoirs, thisprocess conditions the newly recruited monolayer of platelets to becomethe reactive surface for continuing platelet accrual.

Immediately after platelet arrest, the clotting process begins by theparticipation of platelet released substances and fluid phasecoagulation factors. Initiation of the coagulation cascade results inthe conversion of prothrombin to thrombin (a serine protease). Thrombincleaves two pairs of peptides (fibrinopeptides A and B) from theaminoterminal ends of the Aα and Bβ chains of the fibrinogen molecule.Cleavage of fibrinopeptide-A is sufficient to initiate clot assembly(4). The monomer units formed initiate a self-assembly process offorming protofibrils. Weak lateral interactions between protofibersincrease as the protofibers lengthen, resulting in their alignment andcoalescence, to ultimately yield fibers. This process leads to theformation of a network composed of fibrin polymers and spaces filledwith fluid. Once the fibrin network is formed, the platelets begin tocontract, resulting in a pull on the strands of the fibrin network.Platelet contraction requires active restructuring of the plateletcytoskeleton.

Dynamic rearrangements in the cytoskeleton are crucial during plateletactivation in both, initial platelet adhesion to surfaces (see FIG. 1)and platelet to platelet cohesion. Actin polymerization innon-stimulated platelets is limited by monomer-sequestering proteinssuch as thymosin β4, profilin and barbed end-capping proteins such asgelsolin (5-7). Under these conditions, around 2,000 actin filaments aredistributed in the cytoskeleton and in the membrane skeleton right underthe inner surface of the plasma membrane (8). After the stimulation bystrong agonists, there is a rapid increase in actin polymerization, withreorganization of the two actin networks, resulting in a change of shapewith formation of filopodia and lamellipodia at the cell periphery. Thisis followed by redistribution of actin and other cytoskeletal andsignaling proteins form the membrane skeleton to the cytoskeleton(9;10). Platelet spreading is associated with the appearance of actinstress fibers and focal-adhesion-like structures that contain clustersof integrins and vinculin (11). Small GTPases of the Rho family—such ascdc42Hs, Rac, and Rho—have been implicated in the formation offilopodia, lamellipodia and focal adhesion plaques in many cell types(12), and the same may occur in platelets.

Under normal conditions, the coagulation system remains in a finebalance. Pathologic alterations of the system may induce a risk ofhemorrhage or increase the potential for thrombosis. An example of theformer would be the bleeding disorder of hemophilia, which results froma low activity of Factor VIII, a blood clotting protein. An example ofthe latter would be recurrent venous thrombosis in individuals who havedecreased levels of the coagulation inhibitor antithrombin III. Patientswith decreased ability to remove clots, decreased fibrinolyticpotential, are also at risk for thrombosis

To counterbalance the above mentioned mechanisms that precipitateplatelet activation, platelets are downregulated by the anti-thromboticpotential of normal endothelial cells, in vivo, and by substancesproduced by the activated platelets. The majority of pathways thatresult in inhibition of platelet aggregation and procoagulant activitiesact by increasing the internal level of cyclic AMP, which activates thecyclic AMP-dependent protein-kinase. This leads to serine-threoninephosphorilation of an array of substrates.

Experimentally, the result of the platelet contraction and the tensionapplied on the fibrin network strands is observed in vitro as clotretraction. Macroscopically, clot retraction is seen as a dramaticreduction in clot volume in a process that expels the fluid trappedinside the clot. Although the physiological role of clot retraction isnot completely understood, it is assumed that it helps in approximatingthe edges of a tissue defect and in concentrating the clot in the areaof injury (4). One issue that it is clear in clot retraction is that inorder for this process to occur normally all haemostatic mechanisms mustact in synchrony. The two primary requirements for proper clotretraction to occur are the formation of an appropriate fibrin networkand healthy platelets, capable of contracting and anchoring the fibrinnetwork. The structure and formation of the fibrin network are sensitiveto pH, ionic strength, calcium concentration, plasma proteins, plateletrelease products, leukocyte materials, etc. (4).

Examples of pathological conditions that affect the structure of thefibrin network are diabetes mellitus and multiple myeloma (4;13).Healthy platelets need to express the integrin α_(IIb)β₃ on theirsurface to properly anchor the fibrin strands and they need to bemetabolically fit for the task. Examples of pathological conditions thataffect platelet metabolism are diabetes mellitus and uremia (14). Also,the age of platelets affects their performance, this aspect isparticularly important for transfusion purposes.

The wide spectrum of processes involved in clot retraction, includingbiochemical, biorheological and biomechanical mechanisms, in conjunctionwith fine controlling and orchestration mechanisms, makes clotretraction a very desirable focus point that represents the well-beingof all the steps required for this event to take place. This approach of“funnel detection” yields excellent means for population screening andindividual patient monitoring for clinical progress.

Current State-of-the-Art

In clinical practice, the measurement of platelet viability has beenused mainly to set standards for appropriate storage and handling ofplatelet concentrates. These techniques include estimation of thelife-span after storage with radiolabeling, measuring the reduction ofbleeding time, and semi-quantitative estimation of platelets to formaggregates in vitro with the use of platelet aggregometers. Thesetechniques, however, are not routinely used to evaluate plateletperformance. A more practical estimation of the capacity of platelets tofunction normally appears to be retention of shape, ATP content andfunction in the osmotic reversal reaction (15).

Methods Currently Used to Evaluate Clot Retraction

A common method utilized to evaluate clot retraction is quantitation ofthe fluid volume expelled by the clot during retraction, and estimationof the volume of the residual clot (16). This is a qualitative essaythat does not provide information about the force generated during clotretraction.

Another known method involves the formation of cylinders or strips ofclots, which are then immobilized on one end and anchored to a forcetransducer (17) on the other. This technique requires mechanicalmanipulation of the sample and bathing of the clots in a foreignsubstance that may alter the natural process of clot retraction.

Yet another technique utilizes a rheometer to measure the normal forcedevelopment during clotting and retraction (18). An important limitationof this technique is the high cost of the equipment.

A method described by Carr in U.S. Pat. Nos. 4,986,964; 5,293,772 and5,205,159 directly measures the force developed by platelets during clotretraction. Carr's apparatus consists of a cup in which the fluid sample(before clotting) is placed. The opening of the cup is covered by anupper plate, which is coupled to a steel arm attached to a forcetransducer. As the clot retracts, the force generated is transmitted tothe force transducer, where it is measured (1;4;13;14). Although this isa very reliable method, the cost per measurement is high, because thismethod allows only the measurement of one sample at a time. Also, thisequipment has the added complication of the high precision required forits alignment and setup.

Therefore, it would be advantageous to have a low-cost, reliable methodfor the quantitation and monitoring of the force developed during clotretraction. This would provide a simple way to assess several variablesof clinical relevance that converge into one single measurable variable,i.e. using the above mentioned funnel detection philosophy. Moreover,what is needed is an easy-to-use and economical device to accuratelymeasure the force developed during clot retraction. This device shouldbe self-contained in order to minimize exposure to biohazardousmaterials. Such a device would have a broad spectrum of clinicalapplications, including, for example, patient evaluation and populationscreening for pathological conditions.

SUMMARY OF THE INVENTION

The primary object of this invention is to provide a novel plateletretractometer that will measure the force developed by platelets duringblood clot retraction.

Another object in accordance with the present invention is a device thatcan automatically measure the force developed by platelets during clotretraction, is easy and inexpensive to operate, and provides no exposureof the operator to biohazardous materials.

A further, most preferred object is to provide a method for measuringthe force developed by platelets during clot retraction, as well as itsclinical applications, in both research and patient monitoring. Theclinical applications comprise all conditions in which plateletviability and platelet metabolism are impaired, including, but notlimited to, diabetes mellitus, chemotherapy, and monitoring of plateletaging for blood transfusion. In experimental applications, the devicewill increase the scope of study in platelet biology by bringing auser-friendly ready-to-use method useful to dissect the mechanismsinvolved in platelet contraction in both, physiological and pathologicalconditions.

In accordance with these objects, this invention contemplates anapparatus for measuring blood platelet contractility, hereinafter calleda “retractometer.” The retractometer has a spherical rigid chamber withan opening in its dorsal aspect. Found inside this chamber is a smaller,spherical, flexible membrane chamber concentrically aligned and isolatedfrom the larger rigid chamber, creating a void space between the wallsof the rigid and flexible chambers. The flexible membrane chamber alsohas an opening in its upper aspect, smaller than and coaxial to theopening in the rigid chamber. There is a tube attached at the opening,leading out of the flexible chamber concentrically and in perpendicularaxis through the opening in the rigid chamber. This concentric alignmentof chambers creates a void space that is isolated from the void space ofthe flexible inner chamber. A second tubular passage is connected to thevalve at one end and in perpendicular alignment to the first passage. Apressure transducer is connected to the distal end of this second tube.Thus, any force exerted on the flexible chamber to alter its diameterwould be measured by the pressure transducer. The membrane chamber canbe manufactured from latex by dipping a mold and withdrawing a thinspherical bag with an opening created by a shaft attached to a sphericalmold. The flexible membrane can be latex or any other suitable material.

In further accordance with these objects, this invention contemplates analternative retractometer having similar spherical rigid and flexiblechambers, and openings in their upper aspect isolating the two chambersfrom each other and creating a void space between their walls. Thevariation from the above described setup is that the tubular chamberleading out of the flexible chamber concentrically and in perpendicularaxis through the opening in the rigid chamber, has both ends sealed.This creates a void space that is isolated from the void space of theflexible inner chamber. Through this tubular chamber, runs a glasscapillary tubing, coaxial to and longer than the tubular chamber,passing through both ends of the sealed tubular chamber. This creates acontinuous passage from outside of the apparatus to the void space ofthe inner flexible chamber. The distal opening of the capillary tubingis plugged before directly reading the force applied by the retractingclot as the height of the fluid column inside the capillary. This plugcan either be a removable type, like a cap or stopper, or a sealed typethat is opened by breaking the capillary at an etched or scored pointabove the sealed tube.

The advantage of this embodiment is that direct readings can be taken,with no need for electronic measuring equipment. The disadvantage isthat it does not readily lend itself to automation except by opticalreadings.

A more specific and preferred embodiment of this invention is anautomated system for measuring blood platelet contractility of aplurality of samples having an array of retractometer units with valvesas described hereinabove. Each unit retractometer is a separateapparatus for measuring blood platelet contractility of a single sample.As described above, it comprises a spherical rigid chamber having anopening in its upper aspect, a smaller, spherical, flexible membranechamber placed concentrically within the rigid chamber, creating a voidspace between the walls of the rigid and flexible chambers, and havingan opening in its upper aspect that is smaller than and coaxial to theopening in the rigid chamber. A first, attached contiguous tubularpassage leads out of the flexible chamber concentrically and inperpendicular axis through the opening in the rigid chamber, creating avoid space that is isolated from the void space of the flexible innerchamber. A two-way valve is attached to the distal end of the tubularpassage, which in turn, is connected to a second tubular passage. Theend distal to the valve is connected to a common pressure transducer.The valves are operated automatically by solenoids, energized andregulated by an electronic circuit. This circuitry is programmed tochoose and operate the solenoid valves in a predetermined order writtenin software by the inventors.

An equally preferred embodiment in accordance with this invention is anelectronic solenoid valve controller to fully automate a systemcomprising a number of retractometers and save costs in labor. Thisembodiment is a system apparatus for automatically measuring plateletcontractility in a plurality of samples. The system has a pumpmechanically connected to a pump motor, which in turn is electronicallyconnected to a microprocessor having a plurality of pins. One pin isused to turn the pump motor on; a second pin to move fluid in the pumpin one direction; a third pin to move fluid in the pump in an oppositedirection; and at least one of the remainder of the pins to activateeach of an array of solenoid valves. The system also has a voltagedivider used to establish the position of the fluid in the pump. Thereis a fluid conduit connecting the pump to a hydraulic system having amanifold that connects the pump to each of a plurality of retractometerscontrolled by solenoid valves. Each retractometer communicates with oneof the solenoid valves. A pressure transducer reads each pressure andsends the reading to an analog to digital (A/D) converter connectedelectronically to the tranducer, the pump motor, the microprocessor, anda computer.

Basically, sequence of events is as follows. A readout position voltagefrom the voltage divider is entered through the (A/D) converter to themicroprocessor, which determines the direction of flow in the pump andactivates the pump to adjust the fluid pressure within the system. Thepressure is then measured by the pressure transducer connectedelectronically to the A/D converter, and a target pressure is registeredin the microprocessor memory, which is subsequently recorded anddisplayed by the computer.

This apparatus embodiment may have a pump that moves fluid with asliding piston. Preferably, the pump is a syringe controlled by a stepmotor with very fine gradations. Preferably, the apparatus has an arrayof eight solenoid valves, each valve communicating with one of eightretractometers. More preferably, the system apparatus is expandable byaddition of retractometers and valves. Most preferably, theretractometers are packaged in a cartridge, such that one cartridge isremoved after sampling and replaced with another having additionalsamples, and so on.

The apparatus is protected by a protection valve located at the entranceto the pressure transducer to prevent damage to the system and a secondprotection valve to control access to a fluid reservoir. The subroutinesin the analysis program are burnt into the microprocessor.

A most preferred embodiment in accordance with this invention is amethod for measuring blood platelet contractility. The method comprisesthe steps of preparing a retractometer according to this invention byapplying adhesive to the surface of the inner flexible membrane to avoidslippage of clots. The adhesive can be any suitable substance, forexample, collagen Type I suspension. The coated flexible membrane isthen pressure conditioned by mounting it on a rubber stopper pierced bya hypodermic type needle attached to a two-way valve. A syringe isattached to one opening of the valve and a second needle is attached toa second opening of the valve, making certain that the reach of the twoneedles is identical.

Next, the membrane chamber is slightly pressurized, the valve to thesyringe is closed, communication is opened to ambient fluid. In thismanner, the inner and ambient pressures are allowed to equilibrate bysiphoning. The fluid level inside the capillary is adjusted to “zeropressure” level.

The second step involves loading of the sample into the void createdbetween the two chambers, surrounding and in contact with the outsidesurface of the flexible membrane chamber. A small amount of oil is addedover the sample to avoid drying out. The sample is then allowed, orinduced, to clot and the force of the clot retraction is measured in apressure transducer and recorded.

Also contemplated by this invention is a method for automaticallymeasuring a number of samples in a number of retractometers to determinethe strength of platelet contractility. A first step requirescalibration of the apparatus above. This entails the microprocessorreading all initial pressures in all retractometers sequentially byopening each solenoid valve, opening the protection valve, measuring thevoltage in the pressure transducer and storing the measured value in thetemporary memory of the microprocessor. This process is repeated untilall the initial pressure values are registered as target values for eachof the retractometers. The second step adjusts the value of thehydraulics by opening the protection valve only and activating the pumpuntil the target value is reached. The sample is then loaded into theretractometers, and clot formation is induced. The third step requiresopening of the sample valve, measuring the pressure, and closing thesample valve, in that sequence. The measured values are then sent to atext file in a computer, and the new measured value for eachretractometer becomes the next target value. This third step is repeateduntil all samples are measured. The entire process of measuring theclotted samples takes less than one minute.

The methods described here are useful in determining platelet activity.The ability to determine platelet activity, and contractile strength, ismore specifically useful in determining viability of stored bloodproducts. Determining forces of contractility is particularly useful indiagnosis or prognosis of various diseases in patients. Because each ofthe components associated with clotting is the result of a myriad ofintermediate steps, clot retraction is an excellent candidate for“funnel detection” where with one simple measurement it is possible tounmask a vast array of pathological stages. Funnel detection methods areparticularly important in population screening studies.

Still further embodiments and advantages of the invention will becomeapparent to those skilled in the art upon reading the entire disclosurecontained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Platelet interaction with surface immobilized collagen typeI. RICM images show platelets in a flow field. On a gray scale, blackindicates a distance from the surface of 4-12 nm; white of >20-30 nm.The time after initiation of the experiment is shown at the right sideof each panel. For this experiment the platelet count was reduced to10,000 platelets per μl.

FIG. 2 shows the evolution of an isolated thrombus at 100 s⁻¹. For theexperiment shown here, development of a single thrombus was recorded.This image shows the thrombus once it is developed (>10 minutes) toobserve the grow changes. Each image corresponds to the summation of aseries of confocal images. Of notice is the peculiar growth pattern ofthe thrombus. Platelet deposition appears to occur in the downstreamareas.

FIG. 3 is a graphic representation of the STL file generation. For theexperiment shown in this figure, an isolated thrombus obtained withcollagen spray was used. The wall shear rate was 100^(s−1) and the datapresented correspond to images taken after 10 minutes of flow. Thegeometry reconstructed here corresponds to the thrombus shown in theearly time of FIG. 2

FIG. 4 is a photograph of the sterolithography model. The actual modelwas built to a scale of 300:1 and has a volume of 3.44 cm³.

FIG. 5 is a diagrammatic representation of two alternative designs forthe retractometer of this invention. Top panel (A) is a design in whichthe individual retractometers are connected through a system ofcommunicating vessels sharing a common pressure transducer. This designallows the simultaneous measurement of several samples. Bottom panel(B), is an alternate design having a clay plug closing the air filledcapillary tube, which the operator “snaps” by bending it around theetching before starting the reading. The fluid inside the capillary thenreaches the “zero level” corresponding to the hydrostatic pressure ofthe system. This design allows the direct measurement of the pressurewithout the need of electronics.

FIG. 6 is a diagram showing the contracting element. The upper panel ofthe figure represents the fibrin network before platelet contraction.

FIG. 7 diagrammatically represents the origin of the forces developed byplatelet contraction within a retractometer of this invention. When thefibrin network contracts, tension develops along the surface of theelement, due to the “pull” between the contracting elements.

FIG. 8 is a force analysis of the retractometer. A is a diametricalcross-section in a plane passing through the center of the sphere. B isa geometrical representation of two arbitrary but symmetrical vectorsacting on the unit represented in A.

FIG. 9 shows the mechanical model comprising a cantilever beam clampedat one end, subject to a constant bending moment.

FIG. 10 shows the calibration graph of a single cantilevered transducer.Steps of 1 gram were used in the calibration. The means of theexperimental points are shown with their corresponding standard error ofthe mean. The continuous line corresponds to the linear regression ofthe measured points. The correlation coefficient calculated isr²=0.9995. The force resolution of the transducer is 2.85×10⁻⁴ gramforce.

FIG. 11 Top: is a photograph of one embodiment of an immersion mold. Theprototype shown here was manufactured from stainless steel. Bottom: is aprototype of the flexible membrane of this invention.

FIG. 12 is a graphic representation of a method used to pressurecondition the membrane.

FIG. 13 depicts the results of a preliminary experiment to show thefeasibility of the proposed methodology. For the experiment shown herethe flexible membrane used was fabricated with latex with a thickness of150 μm. Citrated blood (11 μM Sodium Citrate) was used.

FIG. 14 illustrates the change in shape of the clots when separated fromthe membrane and cut open. The photograph shows a petri dish with threesections of the clot. A digitally enhanced magnification of the threesamples shown is presented for better appreciation of the process.

FIG. 15 is a schematic of the geometry of the cylindrical clot duringcontraction. The contraction of the clot is considered to be isotropic(17). The force F is measured directly by the force transducer. The areaA can be directly calculated from the measurement of the clot diameter.The stress (F) can be estimated from this simple model.

FIG. 16 is a comparison of the two methods used in this description.Both experiments were performed using platelet rich plasma.

FIG. 17 is a schematic diagram of an electronic solenoid valvecontroller useful in simultaneous processing of many samples.

FIG. 18 is a schematic representation of the fully automated systemapparatus that can greatly increase the speed and ease of measuringplatelet contractility in a number of retractometers, each having thesame or different sample. Such a system is highly useful in screeningpopulations and effectiveness of various drugs.

It is stressed that the figures above represent only certain fullytested working examples and do not limit the invention to these preciseillustrations.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Introduction

After in vitro clot formation, the fibrin meshwork entraps virtually allthe serum and the cellular components of blood. Within minutes to hours,the platelets in the clot contract, expelling a very large fraction ofthe serum. This process is known as clot retraction. Although thephysiological relevance of clot retraction is still not fullyunderstood, the fact that platelets are needed for this process to takeplace is well documented (1). There is strong experimental evidence thatsuggests the participation of an actinomyosin contractile mechanism aswell as the involvement of the platelet α_(IIb)β₃ in the process (2;3).

The present invention is based on the rationale that the development ofa reliable method for the study of clot retraction will bring not only auseful tool to elucidate the mechanisms involved in the physiologicalmechanisms, but also an important tool for the monitoring of the overallwell-being of the platelets in a blood sample. This invention willpotentially yield an important diagnostic tool for the monitoring anddetection of pathological states, as well as an easy-to-use tool for themonitoring of platelet viability for transfusion purposes.

Material and Methods

In order to develop the instant invention, certain relevant parametershad to be elucidated. In the examples hereinbelow, are describeddeterminations of platelet activation under certain conditions, as wellas manipulations of clot geometry.

EXAMPLE 1

Perfusion Studies.

Methods

Blood obtained from healthy volunteers was mixed with D-phenylalanyl-L-prolyl-L-argine chloromethyl ketone dihydrocloride (PPACK,93:M) to prevent clotting. The platelet count was adjusted to 10,000/μlto reduce the number of events on the surface and facilitate imageanalysis. Perfusion experiments were conducted in a parallel plate flowchamber at 37° C. (3) using type I collagen fibrils as reactivesubstrate onto glass coverslips. The interaction of flowing plateletswith the surface was evaluated in real time by reflection interferencecontrast microscopy (RICM) using a Zeiss Axiovert 135M microscope. Inthis technique, interference colors indicate the distance between twosurfaces, such as cellular membranes and a substrate coated on glass. Ona gray scale, zero-order black indicates a separation of 4-12 nm, andwhite a distance >20-30 nm (15;19). Experiments were recorded on S-VHSvideotape at the rate of 30 frames per second and analyzed off-line withMetamorph (Universal Imaging) software.

Results

Platelet Interaction with Surface Immobilized Collagen Type I.

The top panel in FIG. 1 (0 minutes) shows the native spheroidal shape ofthe platelets, before activation. A few seconds after the initialplatelet adhesion occurs, the first signs of activation are seen asdramatic shape changes and subsequent adhesion of the platelet membraneto the reactive surface. The other panels (2 and 4 minutes) show theactivation and spreading that two single platelets undergo. Of notice isthe large area that a single platelet can cover. This experiment showsthe large amount of “membrane reservoir” contained by the relativelysmall platelets. After activation, the platelets initiate contraction,resulting in their deformation and clot formation. This phenomenon isnot observed in the photographs shown here because the platelets areattached to a non-deformable surface (glass); however, the plateletsincrease the tension on their membrane due to the above mentionedcytoskeleton rearrangement.

EXAMPLE 2

In order to demonstrate some of the technical capabilities currentlybeing developed in the Inventors' laboratory, a summary of thedevelopment of a technique to create an upscale replica of an actualthrombus is presented below. The geometrical data of the thrombus areobtained with confocal microscopy while the blood is continuouslyflowing as previously reported (20). This technique was developed tostudy the flow field around a thrombus in an upscale chamber. Bymatching the Reynolds number, it is possible to determine the flow pathin the microscopic realm, based on the similarity principle.

The evolution of an isolated thrombus at 100s⁻¹ is depicted in FIG. 2.For the experiment shown here, a single thrombus was recorded frominitiation of the flow. This image shows the growth changes in thethrombus once it is developed (>10 minutes) to 30 minutes. Each image isderived from the summation of a series of confocal image slices. Of noteis the peculiar growth pattern of the thrombus. Platelet depositionappears to occur in the downstream areas.

EXAMPLE 3

Below, Inventors describe their technical solution to create an upscalethree-dimensional (3-D) model of a thrombus based on the informationobtained with confocal microscopy.

As a first step, Inventors decided to investigate the already availabletechniques for 3-D rapid prototyping. A commonly used technique isstereolithography, which uses step-wise planar buildup of the object,based on the solidification of a photoresin by a laser beam. After eachlayer is cured, the object is lowered into a fluid resin pool by adistance equal to the vertical resolution of the system. This techniqueappears to be adequate in view of the complex geometries that it canhandle.

The main practical difficulty in merging confocal microscopy andstereolithography is the lack of compatibility of the data. Confocalmicroscopy renders the data in a series of images (TIFF files in oursystem). These images are represented by a series of pixels with a givengrayscale value, and the images are separated by the distance of theconfocal sections (1 μm in this case). Stereolithography, on the otherhand, uses ASCII files that contain coordinates of the surfacessurrounding the object. This format has the practical advantage ofpossessing the capability to rotate objects to an orientation thatfacilitates the manufacturing process. Inventors developed the series ofsteps that successfully led to the generation of a Stereolithographyfile from the original images of the experimental data.

EXAMPLE 4

Generation of Stereolithography Files From Confocal Data.

The confocal microscopy images obtained were preprocessed with a 3×3median filter, in order to minimize noise originated by the flow. Theimages were then translated into a voxel space with a software packagewritten by Inventors specifically for this purpose. This packagemaintains the relative positions of the measured objects in a cartesianthree-dimensional space. The voxel representation of the microscopicfield was then used with AVS release 5.3 (Application Visual Systems,Inc., Waltham, Mass.) to render the three-dimensional field. Thissoftware package allows the operator to interpolate a surface(isosurface) between contiguous voxels with an intensity above a presetthreshold. This isosurface is comprised of a series of triangles, eachtriangle being a “surface unit.” The coordinates of these surface unitswere then stored in a geometry information file. The geometry file wasthen translated into an “STL” file (standard input format forstereolithography) with a software package written by Inventorsspecifically for this purpose. The STL file contains the coordinates ofthe vortices of the surface units and a normal vector pointing outsideof the body of the object to be materialized. During the fabricationprocess, a scale factor was included to yield the desired dimensions. Agraphic representation of the file generation is shown in FIG. 3.

Graphic Representation of the STL File Generation.

For the experiment shown in this FIG. 3, an isolated thrombus obtainedwith collagen spray was used. The wall shear rate was 100^(s−1) and thedata presented correspond to images taken after 10 minutes of flow. Thegeometry reconstructed here corresponds to the thrombus shown in theearly time of FIG. 2. The upper left panel shows a summation of all theconfocal images obtained from a real thrombus. The upper right panelshows the topographical representation of the thrombus in pseudocolor.The bar on the right shows the color code for the height in micrometers.The lower left panel shows the 3-D representation rendered with AVS, asdescribed above. Although it is possible to orient the geometry to anyposition, a planar view was chosen for easier comparison of the 3-Drepresentation with the original data.

The lower right panel shows a graphic representation of the STL file.The graph shows a wire model of the file, with the orientation identicalto the previous panels. The “hedgehog” appearance is due to normalvectors pointing outside of the body, as described in the method. Forclarity, the normal vectors are shown in blue, and the wire model inwhite. This type of graphic representation is not necessary for theactual fabrication process, but it is useful for error detection in thecreation of the files and overall quality evaluation of the process. Inthe graph, the complexity of the surface and the large number oftriangles necessary to reconstruct such a complex geometry can beappreciated.

FIG. 4 shows the actual model described above built to a scale of 300:1and has a volume of 3.44 cm³. The running time for this sample was 2.3hours, using a FDM 2000 stereolithography machine (Stratasys, OntarioCalif.). The orientation of this photography is similar but notidentical to the orientation shown in FIGS. 2 and 3.

EXAMPLE 4

Experimental Design of the Retractometer

Principle of Operation

The performance of the device of this invention is based on the Laplaceprinciple, in which the tension developed by the contraction of a sheetof platelets during clot retraction is transformed into an increase inpressure inside a semispherical flexible membrane.

The geometry of the proposed device is the following. Let r_(o) be theradius of a spherical container and r_(i) the radius of a concentricalspherical membrane as shown in FIG. 5(A and B), r_(o)>r_(i),h=r_(o)−r_(i); r_(o),r_(i)>>h.

Two Alternative Designs for the Retractometer.

Two alternate embodiments of the retractometer of this invention areshown in FIG. 5AB. Top panel (A), is a design in which the individualretractometers can be connected through a system of communicatingvessels sharing a common pressure transducer. This design allows thesimultaneous measurement of several samples. In FIG. A, prior toclotting, a blood sample is placed inside (2) the rigid reservoir (3).The thickness of the sample at 2 is h=r_(o)−r_(i). A thin layer ofmineral oil (light white oil, Sigma) is placed on top of the bloodsample to avoid evaporation. As tension along the wall of the flexiblemembrane (4) increases due to clot retraction, the pressure inside thetube (1) increases.

Bottom panel (B) is a design in which the operator “snaps” the clay plug(7) of the air filled capillary tube, by bending it around the etching(scoring) (6) before starting the reading. Then the fluid inside thecapillary reaches the “zero level” corresponding to the hydrostaticpressure of the system (details of the filling of the flexible membraneare given hereinbelow). The filling fluid of the flexible membrane maycontain a coloring agent for easier visualization. The presence of theplug (7) prevents both fluid evaporation and changes in the fluid levelby manipulation of the retractometer. This design allows the directmeasurement of the pressure without the need of electronics.

FIG. 6 is a diagrammatic representation of the contracting elementdescribed in the body of the text. The upper object of FIG. 6 representsthe fibrin network before platelet contraction. As an example, FIG. 6shows an isotropic retraction with a longitudinal strain of −0.5. Thestrain g is defined as g=(L−L₀)/L₀, where L is the length at the end ofthe deformation, and L₀ is the initial length. The bottom object showsthe result of the isotropic contraction.

An important consideration in the design and performance of theretractometer device is that when the shell element described in FIG. 6contracts, the only changes that contribute to an increase in thetension in the contracting element are L1 and L2. A contraction in hdoes not contribute to development of tension on the system described inFIG. 5. This concept is detailed in FIG. 7.

FIG. 7 is a diagram representing the origin of the forces developed byplatelet contraction in a retractometer. When the fibrin networkcontracts, tension develops along the surface of the element, due to the“pull” between the contracting elements. Because the outer surface ofthe contracting element is free, the contraction of the element alongthe thickness h results in a decrease in volume and not a modificationof tension on the contracting element. In order to better represent thisconcept, the diagram on the right shows a free body taken from thecontracting element. A change in radius of the cylindrical componentshown results in an increase in tension along the surface as shown,while a change in height (h) does not modify the tension along thesurface of the plate.

In order to calculate the magnitude by which a variation in the tensionof the clot will result in a variation of internal pressure in thedevice shown in FIG. 5, it is helpful to use a free body diagram asshown in FIG. 8.

FIG. 8 represents a force analysis of the retractometer of thisinvention. The flexible membrane of the retractometer is modeled forthis analysis as a perfect sphere. A is a diametrical cross-section in aplane passing through the center of the sphere as shown. The resultantinner pressure Pi is homogeneously distributed on the inner surface ofthe retractometer. The origin of all the vectors is the center of thesphere and all of them have the same magnitude. The dotted line in thispanel shows the arbitrary cross-section where the analysis is performed.

B is a geometrical representation of two arbitrary but symmetricalvectors acting on the unit represented in A. Notice that the horizontalcomponents (parallel to the cross-section shown in A with the dottedline) of these vectors cancel each other, the vertical component (normalto our arbitrary section shown in A) does not cancel by any of thevectors acting on the lower half of the sphere. Therefore, for the forceanalysis, the only vectoral components of the force resultant of Piacting on the surface are normal to the cross section shown in A. Thesevectors act on the area a₁ shown in C, and a₂ is the sectional area ofthe wall of the sphere. D is a free body diagram of a thin slice of thebody shown in C cut by two parallel planes at a small distance apart,one on each side of the center of the sphere. The circumferential stressF is a stress acting on, and normal to, the cross-sectional plane. <F>is the average value of F, which is non-uniform across the thickness ofthe wall. The value of <F> is computed hereinbelow. The vectors on theright side of D show the condition of equilibrium. As explained above,the force acting vertically and downwards (F₂) is computed as thepressure acting on a₁, or Pi(a₁) and F₂=Pi(a1)=Piπr_(i) ². The area ofthe wall of the contractile element is Br_(o) ²−Br_(i) ². The resultanttensile force due to clot retraction in this particular geometry isF₁=B(r_(o) ²−r_(i) ²)<F>. The balance of the forces in equilibriumrequires, therefore, that F1=F2, or: $\begin{matrix}{{{\pi\left( {r_{o}^{2} - r_{i}^{2}} \right)}\left\langle \sigma \right\rangle} = {\pi\quad r_{i}^{2}P_{i}}} & \left( {D\text{:}1} \right) \\{{or}\text{:}} & \quad \\{\left\langle \sigma \right\rangle = {{P_{i}\frac{r_{i}^{2}}{r_{o}^{2} - r_{i}^{2}}} = \frac{r_{i}^{2}P_{i}}{h\left( {r_{o} + r_{i}} \right)}}} & \left( {D\text{:}2} \right)\end{matrix}$Therefore, the average tensile force can be easily calculated based onthe measurement of the hydrostatic pressure inside the compartmentdefined by the flexible membrane.

This analysis is exact for a perfect sphere. Although the retractometerdeviates from this uniform stress field in the area where the flexiblemembrane attaches to the capillary tube or connecting tube, the stressanalysis is an excellent approximation at the operational level and itis valid for the purposes of the design presented in this description.

In order to be able to calibrate their retractometer and compare it withother known experimental models, Inventors decided to implement a systemdescribed by others (17), in which a cylindrical clot is immersed inice-cold buffer to prevent platelet contraction. The clots are thenanchored and held vertically to the bottom of the container at theirlower end and to a force transducer to the upper end of the clot.

Force Transducer:

An isotonic force transducer was implemented. The system is based on thesingle supported beam principle. The mechanical model is a cantileverbeam clamped at one end, subject to a constant bending moment. Accordingto the following description:

The equation that dictates the behavior of the beam is: $\begin{matrix}{\frac{\mathbb{d}^{2}y}{\mathbb{d}x^{2}} = {\frac{1}{EI}{M(x)}}} & \left( {D\text{:}3} \right)\end{matrix}$where: M is the bending moment imposed by the load on the cantileveredbeam, E is Young's modulus, I is a property of the cross-sectionalgeometry of the beam, the term on the left is the deflection of the beam(assuming a deflection much smaller than the length of the beam).

The deflection y(x) can be calculated by using: $\begin{matrix}{{{{EI}\frac{\mathbb{d}^{2}y}{\mathbb{d}x^{2}}} = M},{{{EI}\frac{\mathbb{d}^{3}y}{\mathbb{d}x^{3}}} = S}} & \left( {{D\text{:}4},5} \right)\end{matrix}$where S is the transverse shear. The bending moment and the transverseshear are related to the lateral load by: $\begin{matrix}{{\frac{\mathbb{d}M}{\mathbb{d}x} = S},{\frac{\mathbb{d}S}{\mathbb{d}x} = w}} & \left( {{D\text{:}6},7} \right) \\{\frac{\mathbb{d}^{2}y}{\mathbb{d}x^{2}} = {\frac{1}{EI}{M(x)}}} & \quad\end{matrix}$

Where w is the lateral load per unit length.

Because a small curvature is assumed, and the slope of the deflection isfinite, the equation to be used instead of D:3 is: $\begin{matrix}{{\frac{\mathbb{d}^{2}y}{\mathbb{d}x^{2}}\left\lbrack {1 + \left( \frac{\mathbb{d}y}{\mathbb{d}x} \right)^{2}} \right\rbrack}^{- \frac{3}{2}} = \frac{M(x)}{EI}} & \left( {D\text{:}8} \right)\end{matrix}$

Integration of equation D:8 yields: $\begin{matrix}{{y(x)} = {{\frac{M}{EI}\frac{x^{2}}{2}} + {Ax} + B}} & \left( {D\text{:}9} \right)\end{matrix}$

Therefore, for a load imposed at a fixed distance in x, for a constantbending moment, and for a small deflection, the deflection is a linearfunction of the moment.

For the implementation of the force transducer, Inventors used aborosilicate glass rod, with a length of 15 cm and a diameter of 1 mm.Due to the relative length of the rod, deflections up to a maximum of 2cm can be considered small.

The results of the calibration experiments are shown in FIG. 10. Therange tested was from 0 to 5 gram force. This range proved to beadequate for the experimental conditions. FIG. 10 shows the calibrationgraph of a single cantilevered transducer that is part of thisinvention. Steps of 1 gram were used in the calibration. The means ofthe experimental points are shown with their corresponding standarderror of the mean. The continuous line corresponds to the linearregression of the measured points. The correlation coefficientcalculated is r²=0.9995. The force resolution of this transducer is2.85×10⁻⁴ gram force.

Results

EXAMPLE 5

Fabrication of the Retractometer

Fabrication of the retractometer required research and development inthe following areas:

-   -   1) Manufacturing of the flexible membranes. Which is        subsequently divided in two steps:    -   a) Fabrication of a suitable immersion mold    -   b) Fabrication of the membranes    -   2) Filling of the membranes. This step is necessary to assure        that the internal pressure of the membranes corresponds to the        hydrostatic pressure of the fluid around them, during operation.    -   3) Adjusting of the fluid level inside the capillary at “zero        pressure” level. This step is necessary for the operator to see        the fluid level above the capillary holders.    -   4) Calibration of the system and comparison with an alternative        method. The alternative method will be to measure directly the        force developed by a cylindrical clot made with platelet rich        plasma of the same donor to serve as our “gold standard.”

Fabrication of a Suitable Immersion Mold

The first step in manufacturing of the flexible membrane is thefabrication of a suitable immersion mold. In a preliminary phase,Inventors fabricated a prototype mold from stainless steel. Turning nowto FIG. 11, the top panel is a photograph of the stainless steelimmersion mold. Although the embodiment shown here was manufactured fromstainless steel, it could likewise be made from any other suitablematerial. The ball has a diameter of {fraction (9/16)}″ and the rod hasa diameter of {fraction (3/32)}″, but these could be of any suitabledimension. The bottom panel shows an embodiment of the flexiblemembrane. This prototype was made to show the feasibility of fabricationusing the immersion mold shown on top. This prototype embodiment wasfabricated using urethane, but any other suitable material could beused. Due to the transparency of the material it is easy to study thethickness of the membrane. The figure shows an even thickness of thematerial along the spherical region of the membrane. However, thisfabrication technique yields an increase in thickness around the regionof the neck (white arrow). It can be concluded from the stress analysisshown in FIG. 8, that the stresses on the wall are uniformly distributedalong the flexible membrane, except around the point of insertion of thecapillary tube. Therefore, it is expected that this thicker region willnot have an important impact in the performance of the retractometer.

Fabrication of the Membranes.

The membranes would preferably be fabricated by experts in dip moldingtechnology, for example by ACC Automation (Akron, Ohio). For thisapplication, their 4-axis dipping system would be particularly suitable.Briefly, the system has 4 axis of operation (vertical, horizontal/palletrotate and form spin), allowing the membrane coating to be uniform alongthe surface of the mold. The equipment has a vertical stroke of 30inches, a vertical axis speed range of 0.01-12 inch/sec, with 0.001inch/sec speed increments. Rotate axis positional range is 1440 degreesin 1 degree positional increments. Rotate axis speed range is 0.1-60degrees/sec in 0.1 degree speed increments. Spin speed range is 10-100RPM in 1 RPM increments. Horizontal axis position is 18 inches in 0.01inch positional increments. Horizontal axis speed range is 0.01-4.0inches/sec in 0.001 inches/sec/Maximum payload capacity of 10 pounds.The membranes are dried in an integrated force air convection electricoven, with programmable temperature control up to 200° C. The forms arespun in the oven to increase drying uniformity.

The membranes are fabricated using two coats of latex without thickeningagent, in a similar fashion to fabricating condoms. Uniformity inthickness and mechanical properties of the membranes are highlyreproducible.

The second step is to develop a suitable method for the filling of themembranes.

Preparation of the Membrane Prior to the Final Assembly of theRetractometer.

In order to assure that the inner pressure of the membrane is inequilibrium with the surrounding fluid, Inventors implemented a simpletechnique shown in FIG. 12. In order to avoid slippage of the clots overthe membrane surface during contraction, the membranes are coated with asuitable adhesive, for example, with a bovine collagen type I suspensionas described elsewhere (2.5 mg/ml in 0.1 M acetic acid) (20). Thismethod gave a firm adhesion of the clot onto latex membranes. It isexpected that membranes of different materials may require otheradhesives.

Membranes are pressure-conditioned as shown in FIG. 12. The flexiblemembrane is mounted on a sealing rubber stop with a needle insertedthrough it. The needle is connected to a two-way stop-cock, which inturn is connected to a syringe and another needle. The reach of bothneedles is the same. In a first step, the syringe is used to slightlypressurize the flexible membrane. In a second step, access to thesyringe is closed and the two needles are allowed to equilibrate theinner membrane pressure and the ambient pressure by siphoning thefluids. This method is reliable in giving zero pressure readings withthe use of a pressure transducer (Validyne DP 15-22, controlled by aValidyne CD379) immediately after inflation.

The third step is the adjusting of the fluid level inside the capillaryat “zero pressure” level. As seen in FIG. 5B, it would be desirable tocontrol the level of the column inside the capillary to make the readingeasier. The height of the column in a capillary tube is dictated by theexpression: $\begin{matrix}{h = {\frac{2\gamma}{\rho\quad g\quad r}\cos\quad\theta}} & \left( {D\text{:}10} \right)\end{matrix}$where, h is the height of the column, γ is the surface tension of thefluid, ρ is the density of the fluid, r is the radius of the capillarytube, and θ is the wetting angle. The pressure inside the flexiblemembrane of the retractometer, can be easily calculated with theexpression. Δp=ρgh.

In order to make the retractometer more user-friendly, it is necessaryto have a good “zero pressure” level inside the capillary, otherwise thereading error may increase. An easy way to do this is by proper choiceof the capillary radius. The possibility of changing the angle of themeniscus θ in equation D:10, with the following method (21).

Hydrophilic Modification of the Capillaries

The glass capillaries are immersed in a 1% (w/w) NaOH water solution.The container is heated to near boiling (bubble formation starting)(approx 90° C.) and incubated for 10 minutes. The solution is removed,and the capillaries are allowed to cool to room temperature. Thecapillaries are then immersed in a 30% (w/w) H₂O₂ solution, and heatedto near boiling (approx 90° C.) for one hour, washed five times withdeionized, demineralized water, tap dried and placed in a drying oven(250° C.) for 12 hours. Column heights were improved from 16 mm (with a0.75 radius, untreated capillary) to 60 mm (with a 0.5 mm radius,hydrophilic modified capillary).

The fourth step is the calibration of the retractometer and comparisonof the results to an alternative, known method.

In order to explore the feasibility of the methodology in the presentinvention, a prototype retractometer was implemented, as detailed inFIG. 5. For the setup of these preliminary experiments, it was decidedto use a latex flexible membrane with a thickness of 150 μm. Thepressure in both experiments was continuously recorded using a pressuretransducer (Validyne DP15-22, controlled by a Validyne CD379). Acitrated blood sample was separated into two aliquots, one aliquot wasused to prepare a platelet-rich plasma sample by centrifugation at 150×gfor 10 minutes. The other aliquot was used directly without enrichment.Prior to beginning the experiment, a sample (platelet rich plasma orblood) was supplemented with calcium to initiate coagulation. A solutionof 0.2 M CaCl₂ at 42 μl/ml of blood and 65 μl/ml of platelet-richplasma, the difference in volumes accounts for the inert volume occupiedby red blood cells in the whole blood sample. During the experiment,samples were kept at 37° C. The results of these experiments are shownin FIG. 13. As predicted, platelet contractility results in an increasein the hydrostatic pressure inside the flexible membrane.

In order to demonstrate that the increase in hydrostatic pressure shownin FIG. 13 was indeed due to an increase in the tension on the fibrinnetwork, the retractometer was disassembled at the end of theexperiment, the membrane and the clot were immersed in a phosphatebuffered saline solution to avoid drying of the sample. The membrane andthe attached fibrin clot were then sectioned in rings parallel to theequator, the rings were attached to ribbons and the clots were carefullyseparated from the latex membrane. The rationale for the cuts was tounveil the residual stresses in the clots. Cutting introduces newsurfaces on which the traction is zero. Cutting an unloaded body withoutresidual stress will not cause strain. If strain changes by cutting,there is residual stress. The results shown in FIG. 13 demonstrate thefeasibility and validity of the principle of operation of themethodology. The stresses along the thickness of the clot are notuniform, due to the geometry of the retractometer. This lack ofuniformity in stresses must, therefore, result in “shearing strain”across the thickness of the clot. This is seen macroscopically in FIG.14 as twisting of the clots. FIG. 14 shows a petri dish with threesections of the clot. A digitally enhanced magnification of the threesamples shown is presented for better appreciation of the process. Itshould be noted that the larger the deformation the larger is theresidual stress. The large twisting deformation is due to thenon-uniform increase in tension along the thickness of the wall of theclots.

An Alternative Method and Comparison of Results

Inventors decided to implement a method described by others (17) forcalibration and comparison. The method is briefly described below.

Cylindrical clots are obtained by pouring a human platelet-rich plasma(PRP) suspension, immediately after thrombin addition, into cylindricalplastic molds (6 mm diameter and 5 cm in length). The molds are pluggedat both ends with plastic plugs. The sides of the molds are slit foreasier clot extraction, but the ends meet in a manner such that dryingout is prevented. After 10 minutes, the clot cylinders are poured into aPetri dish containing ice cold Tyrode solution to inhibit contraction.The clots are then tied at one end with a cotton thread to a rigidstainless steel support and the other end to a force transducer asdescribed hereinabove.

In order to compare the experimental results of clot retraction with thetwo different setups, it is helpful to link the two methods by thestress generated by platelet contraction.

FIG. 15 outlines schematically the geometry of the cylindrical clotduring contraction. The contraction of the clot is considered to beisotropic (17). The force F is directly measured by the forcetransducer. The area A can be directly calculated from the measurementof the clot diameter. The stress (F) can be estimated from this simplemodel.

Assuming that the stress generated by the platelets is the same in thetwo retractometers, it follows from equation D:2 and FIG. 15 that:$\begin{matrix}{\frac{F}{A} = {P_{i}\frac{r_{i}^{2}}{r_{0}^{2} - r_{i}^{2}}}} & \left( {D\text{:}11} \right)\end{matrix}$Regarding the units of both expressions: P_(i) is given in cm H₂0: 1 cmH₂0=1 gf/cm².

Turning now to FIG. 16 for a comparison between the two methods used.Both experiments were performed using platelet rich plasma. In order tocompare the results, data are presented in terms of the stress assuggested by equation D:11. The solid line shows the results obtainedwith the cylindrical clot and the circles represent the experimentaldata points obtained with the method of this invention.

The values calculated by equation D:11 are highly dependent on theaccurate measurement of the radii in both, the cylindrical clot systemand the retractometer of this invention.

Immediately after the mechanical test, the clots are fixed in 1.25%(vol/vol) glutaraldehyde diluted in 0.1M phosphate buffer (pH 7.2) forone hour at room temperature. The clots are then postfixed in 1%(wt/vol) osmic acid containing 1.5% potassium ferrocyanide for one hourat 4° C. Subsequently, they are dehydrated using graded alcohols andpropylene oxide before being embedded in Epon. We have successfully usedthis technique to estimate the ultrastructure of fibrin clot deformation(22). This step is done only for calibration purposes and it is notintended to be used as a routine in the future.

EXAMPLE 6

Described below is an electronic circuit designed to operate theindividual solenoid valves controlling the hydraulics of thecommunicating vessels for the embodiment shown in FIG. 5A.

The electronic solenoid valve controller circuit is shown in FIG. 17.For the circuit shown here, a 10×16 array was implemented. The circuitlabeled digital row selection is meant for an alternative computercontrol. When a word is written to the gain select input of the CMOScircuit shown (analog to CD4066), a voltage is generated at the outputof the circuit, which is used to select the row on the right handcircuits. In principle, the voltage used for row selection can also beselected manually via a potentiometer part of a voltage divider.Implementation of these circuits allows the operator to select the roweither manually or via digital input. The operational amplifier (RowGain) is intended to give the maximal gain of the voltage divider forrow selection. The use of this is to select manually the maximal rownumber to be read in a given cycle. This non-inverting input of theoperation amplifier (op amp) is amplified and sent to an A/D converter,implemented by the comparators and the priority encoder. Should thecontrol be exclusively digital, this part of the circuit is obsolete, inwhich case the already digital input should be sent directly to the 1 of10 decoder (10 is arbitrarily chosen in this case, and the total numberof rows can be different). The final result of this architecture is thatonly one row is activated at a time.

For column selection, Inventors chose to add a timer assuming that thecolumn selection is done in a continuous sweep. The timer shown in thelower left corner of the diagram (FIG. 17) has a feedback loopcontrolled by a potentiometer that allows the operator to control thesweeping rate. The output of the timer serves as the input for a 4-bitcounter, the output of the counter is input into a 1 of 16 decoder toselect only one column at a time.

EXAMPLE 7

Another embodiment of an electronic solenoid valve controller of thisinvention is shown in FIG. 18. For the example shown here, an 8051microprocessor is used. Three pins of the microprocessor actuate asyringe pump. Pin one is used to turn the pump on and the other two tomove the pump piston either up or down. The pump is directly connectedto the hydraulics (retractometers) of the system. The motor of the pumpis connected to a voltage divider that yields a voltage used toestablish the position of the piston of the pump. This readout positionvoltage is entered through an analog to digital (A/D) converter to themicroprocessor. Other pins of the microprocessor are connected to eachone of the solenoid valves used in the array.

In this example, a series of eight valves are used to measure each oneof the retractometer samples and two others are used to provideprotection to the system. One of the protection valves is located at theentrance of the pressure transducer, and its role is to prevent damageto the system due to the operation of the pump. The other valve islocated to provide access to a fluid reservoir. This valve is used inthis example to fill the syringe prior to the beginning of theexperiments. The output voltage of the pressure transducer is enteredinto the A/D converter and subsequently to the microprocessor. For thisexample, the subroutines were burnt into the microprocessor. In aninitial stage of operation, the microprocessor reads all initialpressures of all the samples by opening each individual sample valve,followed by opening of the protection valve. The voltage from thepressure transducer is measured and stored in the temporary memory ofthe microprocessor. This process is repeated until all the initialpressure values are registered.

In the following cycles, the previous pressure value for the valve thatwill be measured is taken as the target value. Then the value of thehydraulics is taken, having only the protection valve opened. The pumpis then actuated (either up or down depending on the relative value ofthe target pressure) until the target pressure value is reached. Thesample valve is then opened, the pressure is measured, and the samplevalve is closed. The measured values are sent to a text file in a PCcomputer via a serial port. The new measured value for each valvebecomes the next target value. These cycles are repeated until the endof the experiment.

While the present invention has now been described in terms of certainpreferred embodiments, and exemplified with respect thereto, one skilledin the art will readily appreciate that various modifications, changes,omissions and substitutions may be made without departing from thespirit thereof It is intended, therefore, that the present invention belimited solely by the scope of the following claims.

References

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1. An apparatus for measuring blood platelet contractility, comprising:a spherical rigid chamber having an opening in its upper aspect; asmaller, spherical, flexible membrane chamber placed within,concentrically and isolated from the rigid chamber creating a void spacebetween the walls of the rigid and flexible chambers and having anopening in its upper aspect smaller than and coaxial to the opening inthe rigid chamber and a first, attached contiguous tubular passageleading out of the flexible chamber concentrically and in perpendicularaxis through the opening in the rigid chamber, creating a void spacethat is isolated from the void space of the flexible inner chamber; atwo-way valve attached to the distal end of the tubular passage; asecond tubular passage connected to the valve at one end and inperpendicular axis to the first passage; and a pressure transducerconnected to the other end of the second passage wherein any forceexerted on the flexible chamber to alter its diameter would be measuredby the pressure transducer.
 2. The apparatus according to claim 1,wherein the flexible membrane is latex.
 3. An apparatus for measuringblood platelet contractility, comprising: a spherical rigid chamberhaving an opening in its upper aspect; a smaller, spherical, flexiblemembrane chamber placed within, concentrically and isolated from therigid chamber creating a void space between the walls of the rigid andflexible chambers and having an opening in its upper aspect smaller thanand coaxial to the opening in the rigid chamber and a tubular chamberleading out of the flexible chamber concentrically and in perpendicularaxis through the opening in the rigid chamber, having both ends sealedcreating a void space that is isolated from the void space of theflexible inner chamber; and a glass capillary tubing coaxial to andlonger than the tubular chamber, passing through both ends of the sealedtubular chamber, creating a continuous passage from outside of theapparatus to the void space of the inner flexible chamber.
 4. Theapparatus according to claim 3, wherein the distal opening of thecapillary tubing is plugged.
 5. The apparatus according to claim 4,wherein the plug is removable.
 6. The apparatus according to claim 4,wherein the capillary tubing outside of the tubular chamber is scored tofacilitate a clean break.
 7. An automated system for measuring bloodplatelet contractility of a plurality of samples, comprising: an arrayof retractometer units, each of which is a separate apparatus formeasuring blood platelet contractility, comprising: a spherical rigidchamber having an opening in its upper aspect; a smaller, spherical,flexible membrane chamber placed concentrically within the rigid chambercreating a void space between the walls of the rigid and flexiblechambers and having an opening in its upper aspect smaller than andcoaxial to the opening in the rigid chamber and a first, attachedcontiguous tubular passage leading out of the flexible chamberconcentrically and in perpendicular axis through the opening in therigid chamber, creating a void space that is isolated from the voidspace of the flexible inner chamber; a two-way valve attached to thedistal end of the tubular passage; a second tubular passage connected tothe valve at one end and in perpendicular axis to the first passage; anda common pressure transducer connected to the other end of the secondpassage of each separate retractometer wherein any force exerted on theflexible chamber of each retractometer to alter its diameter would bemeasured by the common pressure transducer.
 8. The automated systemaccording to claim 7, wherein the valve is activated by a solenoid. 9.The automated system according to claim 7, wherein the array isconnected to an electronic solenoid valve controller.
 10. A systemapparatus for automatically measuring platelet contractility in aplurality of samples, comprising: a pump; mechanically connected to apump motor; electronically connected to a microprocessor having aplurality of pins a first pin to turn the pump motor on a second pin tomove fluid in the pump in one direction a third pin to move fluid in thepump in an opposite direction and at least one of the remainder of theplurality of pins to activate each of an array of solenoid valves; avoltage divider used to establish the position of the fluid in the pump;a first fluid conduit; connecting the pump to a a hydraulic systemcomprising a first manifold; connecting the pump to each of a pluralityof retractometers each activated by one of the solenoid valves; a secondfluid conduit manifold; connecting the retractometers to a a pressuretransducer; an analog to digital (A/D) converter connectedelectronically to the tranducer, the pump motor and the microprocessor;and a computer wherein, a readout position voltage from the voltagedivider is entered through the (A/D) converter to the microprocessor,which determines the direction of flow in the pump and activates thepump fluid pressure within the system, which pressure is then measuredby the pressure transducer connected electronically to the A/D converterand a target pressure is registered in the microprocessor memory andsubsequently recorded and displayed by the computer.
 11. The apparatusaccording to claim 10, wherein the pump moves fluid with a slidingpiston.
 12. The apparatus according to claim 1 1, wherein the pump is asyringe.
 13. The apparatus according to claim 10, wherein the array ofsolenoid valves comprises eight valves, each valve activating one ofeight retractometers.
 14. The apparatus according to claim 10, furthercomprising a first protection valve located at the entrance to thepressure transducer to prevent damage to the system and a secondprotection valve to control access to a fluid reservoir.
 15. Theapparatus according to claim 10, wherein the output voltage of thepressure transducer is entered into the A/D converter and subsequentlyto the microprocessor.
 16. The apparatus according to claim 10, whereinsubroutines are burnt into the microprocessor.
 17. A method formeasuring blood platelet contractility, comprising: preparing aretractometer according to this invention by applying adhesive to thesurface of the inner flexible membrane to avoid slippage of clotspressure conditioning the flexible membrane by mounting the membrane ona rubber stopper having a needle attached to a two-way valve attaching asyringe to one opening of the valve attaching a second needle to asecond opening of the valve, making certain that the reach of the twoneedles is identical; pressurizing the membrane closing the valve to thesyringe allowing the inner and ambient pressures to equilibrate bysiphoning and adjusting the fluid level inside the capillary to “zeropressure” level; loading a sample into a void outside the flexiblemembrane; adding a small amount of oil over the sample to avoid dryingout of sample; allowing the sample to clot; and measuring the degree ofcontractility in a pressure transducer.
 18. A method for automaticallymeasuring a plurality of samples to determine strength of plateletcontractility, comprising: a first step of calibrating the apparatus ofclaim wherein the microprocessor reads all initial pressures in allretractometers sequentially by opening each solenoid valve, opening theprotection valve. measuring the voltage in the pressure transducerstoring the measured value in the temporary memory of themicroprocessor. wherein this process is repeated until all the initialpressure values are registered as target values for each of theretractometers; a second step, wherein the value of the hydraulics istaken opening the protection valve only activating the pump until thetarget value is reached; and a third step of opening the sample valvemeasuring the pressure closing the sample valve sending the measuredvalues to a text file in a computer wherein, the new measured value foreach retractometer becomes the next target value, and wherein step threeis repeated until all samples are measured.
 19. The method according toclaim 17, wherein the contractility is useful in determining plateletactivity.
 20. The method according to claim 18, wherein the plateletactivity is useful in determining viability of stored blood products.21. The method according to claim 18, wherein the contractility isuseful in diagnosis or prognosis of various diseases in patients.